Parathyroid hormone (PTH)-related protein (PTHrP) was first discovered as the causative agent of humoral hypercalcemia of malignancy (HHM) through the interaction of its amino-terminus (N-terminus) with the PTH/PTHrP receptor (PTH1R). Since then, PTHrP has been implicating in regulating bone formation and the commitment of BM stromal cells (BMSC) to the osteoblastic lineage, which has led to the use of the N-terminal domain of PTH in the management of osteoporosis. In the hematopoietic stem cell (HSC) niche, activation of the PTH1R on osteoblasts promotes HSC activity and the application of N-terminal PTH in BM transplantation. While both bone formation and HSC niche regulation are attributed to the N-terminus, no information exists regarding the role of other domains of PTHrP. Since several functions of PTHrP are attributed to the nuclear localization sequence (NLS), we have created a mouse lacking the NLS of PTHrP. The objectives of this proposal are to investigate the role of nuclear PTHrP on bone formation and the skeletal microenvironment, namely the HSC niche, of which three specific aims are proposed. First, determine the role of nuclear PTHrP on osteogenesis in vitro, by measuring indices of osteogenic, adipogenic, myogenic, and chondrogenic differentiation of BMSC, and in vivo by measuring the effects of PTHrP analogs on bone formation. Second, examine the role of nuclear PTHrP on skeletal microarchitecture, protein synthesis, and gene expression by assessing microstructural indices at sites of endochondral and intramembranous ossification, its interaction with bone regulatory proteins, and the expression of bone regulatory genes. Third, determine the role of nuclear PTHrP in the regulation of the HSC niche by measuring the expression of associated genes in BMSC and HSC and enumerating BM HSC. We hypothesize that the NLS is necessary and additive to the effects of N-terminal PTHrP in bone formation and skeletal microenvironment regulation. Findings will warrant investigating the use of NLS-containing PTHrP analogs, which could have significant implications in orthopedics, oncology and hematology. Relevance to public health: PTHrP is a protein similar to PTH, which is the only proven therapy for osteoporosis. Unlike PTH, PTHrP has a region called the NLS, which takes the protein to the cell nucleus and may contribute to PTHrP's more widespread effects. We will investigate the role of the NLS in bone formation and skeletal microenvironment regulation using a mouse lacking the NLS of PTHrP, which may lead to the use of NLS-containing PTHrP analogs as both a skeletal anabolic agent and for HSC expansion in BM transplantation and related disorders.